The present invention relates to a novel enzyme. In particular, the present invention relates to an enzyme which releases an amino acid having a glycated xcex1-amino group.
Proteases are used in various industrial fields. For example, proteases are used for determining a glycated protein, e.g., glycated albumin, in serum, which can serve as a significant index for the diagnosis, treatment, etc. of diabetes.
Such determination of the glycated protein utilizing the protease can be carried out, for example, by degrading the glycated protein with the protease, reacting the resultant degradation product with a fructosyl amino acid oxidase (hereinafter, referred to as xe2x80x9cFAODxe2x80x9d), and then determining oxygen consumption or hydrogen peroxide generation to find the amount of the glycated protein. Examples of the protease include those disclosed in JP 5(1993)-192193 A and JP 7(1995)-289253 A.
The above-mentioned protease pre-treatment of the glycated protein is conducted because FAOD and the like easily act on a glycated amino acid and a glycated peptide whereas they hardly act on the glycated protein itself. Particularly, since the glycated site of glycated hemoglobin (hereinafter, referred to as xe2x80x9cHbA1cxe2x80x9d) is the N-terminal amino acid residue of the xcex2-chain, there has been a demand for a protease capable of treating HbA1c so that FAOD easily can act on this site of the HbA1c.
Therefore, it is an object of the present invention to provide a novel enzyme capable of treating a glycated protein and a glycated peptide so that FAOD easily can act thereon.
First, among various FAODs, the inventors of the present invention have studied the mechanism of the action of the FAOD that acts on a glycated protein, glycated peptide, glycated amino acid, etc. in which a sugar is bound to an xcex1-amino group. From this study, the inventors have found that such FAOD easily acts on the glycated amino acid in which a sugar is bound to an xcex1-amino group whereas it hardly acts on the above glycated protein and glycated peptide. Based on this finding, the inventors have isolated various bacteria from nature, cultured them, and studied the enzymes produced by them. As a result, the inventors have succeeded in isolating the bacteria producing a novel enzyme capable of releasing an amino acid having a glycated xcex1-amino group (xcex1-Glycated Amino acid: hereinafter, referred to as xe2x80x9cxcex1-GAxe2x80x9d) from the glycated protein or glycated peptide in which sugar is bound to an xcex1-amino group (an N-terminal amino group), thereby establishing the present invention. The novel enzyme (xcex1-Glycated Amino acid Releasing Enzyme: hereinafter, referred to as xe2x80x9cxcex1-GARExe2x80x9d) according to the present invention can release xcex1-GA, for example, from the above glycated protein or glycated peptide. Hence, the determination of HbA1c using the FAOD that easily acts on xcex1-GA can be made practical in clinical tests etc. by the use of this novel enzyme. The novel enzyme according to the present invention can be utilized not only for the determination of HbA1c but also in various application fields, e.g., for the determination of other glycated proteins. Furthermore, in addition to the catalytic functions of releasing xcex1-GA, xcex1-GARE of the invention may have other catalytic functions, e.g., the function of cleaving other peptide bonds. Examples of the novel bacterial strains isolated by the present inventors include bacterial strains of the genus Sphingobacterium, the genus Sphingomonas, the genus Comamonas, the genus Mucor, and the genus Penicillium. However, it is to be noted that xcex1-GARE according to the present invention is not limited to those derived from the strains of these genera.
The glycated amino acid released by xcex1-GARE is not specifically limited as long as it has a glycated xcex1-amino group. However, since the N-terminal valine residue is glycated in HbA1c as described above, the glycated amino acid released by xcex1-GARE preferably is a glycated valine (hereinafter, referred to as xe2x80x9cxcex1-GVxe2x80x9d).
Examples of xcex1-GARE according to the present invention include the following five types.
The first xcex1-GARE is derived from the bacterial strain of the genus Sphingobacterium and most preferably from Sphingohacterium mizutae KDK1003. Sphingobacterium mizutae KDK1003 was isolated from the soil novelly by the present inventors. Sphingobacterium mizutae KDK1003 has been deposited with the National Institute of Bioscience and Human Technology (NIBH), Agency of Industrial Science and Technology, Ministry of International Trade and Industry (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki 305-0046, JAPAN) under the Accession Number FERM P-17348 since Mar. 29, 1999. Bacteriological properties of this strain are as shown below.
(Morphological Characteristics)
This strain is a non-motile, rod-shaped bacterium (bacillus) of 0.5xc3x971.2 xcexcm.
(Culture Characteristics)
When cultured in an agar medium according to the usual method, the strain forms a colony that is circular in form, low convex in elevation, with an entire margin. The colony color is yellow.
The second xcex1-GARE is derived from the bacterial strain of the genus Sphingnomonas and most preferably from Sphingomonas parapaucimobilis KDK1004. Sphingomonas parapaucimobilis KDK1004 also was isolated from the soil novelly by the present inventors. Sphingomonas parapaucimobilis KDK1004 has been deposited with the National Institute of Bioscience and Human Technology (NIBH), Agency of Industrial Science and Technology, Ministry of International Trade and Industry (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki 305-0046, JAPAN) under the Accession Number FERM P-17347 since Mar. 29, 1999. The original deposit has been transferred to one under the Budapest Treaty under the Accession Number FERM BP-7041 (Date of acceptance: Feb. 21, 2000) in the National Institute of Bioscience and Human Technology as an international depositary authority. Bacteriological properties of this strain are as shown below.
(Morphological Characteristics)
This strain is a motile, rod-shaped bacterium (bacillus) of 0.8xc3x972.0 xcexcm.
(Culture Characteristics)
When cultured in an agar medium according to the usual method, the strain forms a colony that is circular in form, low convex in elevation, with an entire margin. The colony initially is translucent and then turns to light yellow. When cultured in the Mac Conkey""s culture medium, the strain does not grow. Further, at a culture temperature of 42xc2x0 C., the strain grows, albeit weakly.
The third xcex1-GARE is derived from the bacterial strain of the genus Comamonas and most preferably from Comamonas acidovorans KDK1005. Comamonas acidovorans KDK1005 also was isolated from the soil novelly by the present inventors. Comamonas acidovorans KDK1005 has been deposited with the National Institute of Bioscience and Human Technology (NIBH), Agency of Industrial Science and Technology, Ministry of International Trade and Industry (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki 305-0046, JAPAN) under the Accession Number FERM P-17346 since Mar. 29, 1999. Bacteriological properties of this strain are as shown below.
(Morphological Characteristics)
This strain is a motile, rod-shaped bacterium (bacillus) of 0.6xc3x971.5 xcexcm.
(Culture Characteristics)
When cultured in an agar medium according to the usual method, the strain forms a colony that is circular in form, convex in elevation, with rhizoids. The colony color is shiny yellow.
The fourth xcex1-GARE is derived from the bacterial strain of the genus Mucor and most preferably from Mucor circinelloides f. janssenii KDK3004. Mucor circinelloides f. janssenii KDK3004 also was isolated from the soil novelly by the present inventors. Mucor circinelloides f. janssenii KDK3004 has been deposited with the National Institute of Bioscience and Human Technology (NIBH), Agency of Industrial Science and Technology, Ministry of International Trade and Industry (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki 305-0046, JAPAN) under the Accession Number FERM P-17345 since Mar. 29, 1999. Bacteriological properties of this strain are as shown below.
(Morphological Characteristics)
This strain forms subglobose sporangiospores of 4 to 6 xcexcm diameter. The sporangia are black and about 70 xcexcm in diameter, and collarettes are observed at the substrate thereof. The width of each hypha is 10 to 12 xcexcm. No rhizoid is formed.
(Culture Characteristics)
When cultured in an agar medium according to the usual method, the strain forms zygospores only by the gametangial copulation. The aerial hyphae with a cotton-wool-like shape initially are white and turn gray gradually. The rear face of the colony is white. The strain does not grow well at 37xc2x0 C. and does not grow at all at 40xc2x0 C.
The fifth xcex1-GARE is derived from the bacterial strain of the genus Penicillium and most preferably from Penicillium waksmanii KDK3005. Penicillium waksmanii KDK3005 also was isolated from the soil novelly by the present inventors. Penicillium waksmanii KDK3005 has been deposited with the National Institute of Bioscience and Human Technology (NIBH), Agency of Industrial Science and Technology, Ministry of International Trade and Industry (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki 305-0046, JAPAN) under the Accession Number FERM P-17344 since Mar. 29, 1999. Bacteriological properties of this strain are as shown below.
(Morphological Characteristics)
Conidiophores rising from the substrate are smooth and covered with thin cell walls and form no terminal vesicle. The conidiophores do not branch. Two to five metulae forming verticils are developed. A single phialide of 0.9 xcexcm is formed at the tip of each metula. The shape of the phialide is like a flask having a tapered short neck. The conidiophores form catenulate, substantially spherical conidiophores of 3 xcexcm diameter, which are covered with smooth cell walls.
(Culture Characteristics)
When cultured in an agar medium according to the usual method, the strain forms a colony that becomes 30 mm in diameter by the culture peroformed at 25xc2x0 C. for 7 days. The colony has a felt-like green surface. The hyphae exhibit no color. The rear face of the colony is light blue. The strain produces no leachate. Growth is negative when cultured at 37xc2x0 C.
A method of determining a glycated protein or a glycated peptide according to the present invention includes: degrading a glycated protein or a glycated peptide with an enzyme; causing a redox reaction between the resultant degradation product and FAOD; and determining the redox reaction so as to determine the glycated protein or the glycated peptide. In this method, the novel enzyme (xcex1-GARE) according to the present invention is used as the above enzyme. The type of the xcex1-GARE used in this method is decided appropriately depending on the type, concentration, etc. of the glycated protein or glycated peptide to be determined. The xcex1-GARE may be used alone or in combination of two or more types. The glycated protein or the like may be degraded by the pretreatment with another enzyme (e.g., protease) so that the xcex1-GARE more easily can act thereon.
In the determination method according to the present invention, the redox reaction preferably is determined by measuring the amount of hydrogen peroxide produced by the redox reaction or the amount of oxygen consumed by the redox reaction. The amount of the hydrogen peroxide preferably is measured using a peroxidase and a substrate that develops color by oxidation (hereinafter, referred to as xe2x80x9cchromogenic substratexe2x80x9d). The amount of the hydrogen peroxide can be determined not only by the above-mentioned enzymic method utilizing the peroxidase or the like but also by an electrical method, for example.
Examples of the chromogenic substrate include N-(carboxymethylaminocarbonyl)-4,4xe2x80x2-bis(dimethylamino)diphenylamine sodium (for example, the trade name xe2x80x9cDA-64xe2x80x9d available from Wako Pure Chemical Industries, Ltd.), orthophenylenediamine (OPD), and a substrate obtained by combining a Trinder""s reagent and 4-aminoantipyrine. Examples of the above Trinder""s reagent include phenols, phenol derivatives, aniline derivatives, naphthols, naphthol derivatives, and naphthylamine, naphthylamine derivatives. Further, in place of the above aminoantipyrine, it is possible to use aminoantipyrine derivatives, vanillin diamine sulfonic acid, methyl benzothiazolinone hydrazone (MBTH), sulfonated methyl benzothiazolinone hydrazone (SMBTH), and the like. Among these chromogenic substrates, N-(carboxymethylaminocarbonyl)-4,4xe2x80x2-bis(dimethylamino)diphenylamine sodium is particularly preferable.
In the determination method according to the present invention, a sample to be analyzed preferably is blood cells because determining HbA1c in blood cells is useful for diagnosis of diabetes as described above. However, the sample is not limited to the blood cells because blood components other than the blood cells (whole blood, plasma, serum, etc.); biological samples such as urine and spinal fluid; beverage such as juice; food such as soy sauce and sauce, etc. also contain glycated proteins. Also, an analyte to be determined is not limited to HbA1c and can be, for example, a glycated protein such as gelatin and casein or a glycated peptide.
A kit for determining a glycated protein or a glycated peptide according to the present invention includes a protease, FAOD, a peroxidase, and a substrate that is oxidized through a reaction with the peroxidase. In this determination kit, the protease contains an xcex1-GARE of the present invention. The determination method according to the present invention can be carried out rapidly and easily by using this kit. Also in this determination kit, the xcex1-GARE may be used alone or in combination of two or more types.
In the determination kit according to the present invention, as the substrate to be oxidized, the chromogenic substrates described above preferably are used. Further, an analyte and a sample to be used in this kit also are as described above.
A method of producing xcex1-GARE of the invention includes the step of culturing a novel bacterial strain of the invention. This method enables easy production of xcex1-GARE according to the present invention.
It is preferable that the method of producing xcex1-GARE of the present invention further includes the following purification steps (a) to (c):
(a) the step of removing the bacterial cells from the culture solution to prepare a supernatant;
(b) the step of precipitating the protein contained in the supernatant with ethanol; and
(c) the step of separating the protein by chromatography.
The above purification steps are not specifically limited and may be performed along with other purification steps. Further, it is possible to perform, for example, the step (a) only; to perform the two or more steps; or to perform the same step repeatedly.
The xcex1-GARE of the invention obtained through the above-mentioned culture of the bacterial strain may be used in the state of being contained in the culture solution or in the state of being purified. The xcex1-GARE can be used regardless of its purification level as long as it can release xcex1-GA from the glycated protein or the like. However, if purified, a specific activity of the xcex1-GARE can be improved because the components other than the xcex1-GARE in the culture solution are removed by the purification. Accordingly, the amount of the xcex1-GARE to be used can be reduced, which facilitates the handling of the xcex1-GARE. In addition, when the xcex1-GARE is used for causing various reactions, influence given by the components other than the xcex1-GARE can be avoided.
The genes encoding xcex1-GARE of the present invention preferably are prepared by determining the amino acid sequence and the gene sequence of the xcex1-GARE purified through the above purification steps. The genes encoding xcex1-GARE of the invention are not limited to those necessary for the expression of the xcex1-GARE. Examples of such genes include a DNA fragment, RNA, etc., which are used as a probe or primer, and a fragment synthesized chemically based on the gene sequence of the above xcex1-GARE. Using such genes, a recombinant or the like may be prepared to produce xcex1-GARE. The genes of xcex1-GARE according to the present invention can be obtained, for example, in the following manner.
First, xcex1-GARE of the invention is purified, for example, by the purification steps described below. Then, the amino acid sequence thereof is determined by the usual method such as Edman degradation, from which the gene sequence of the xcex1-GARE is assumed. Then, based on the gene sequence thus obtained, a DNA fragment, RNA fragment, or the like is produced by the usual method such as chemical synthesis or the like. Then, the genes of the xcex1-GARE of the invention can be obtained by cloning the genes encoding the xcex1-GARE from a novel bacterial strain of the invention using the DNA fragment, RNA fragment, or the like as a primer, probe, or the like.